Hypoxic stress responses are essential for cellular and organismal survival and drive gene regulation across diverse biological pathways, including cell cycle progression and energy metabolism. Here, we show that topoisomerase IIβ (TOP2B) regulates DNA topology and transcription of hypoxia-inducible genes (HIGs) in a DNA-dependent protein kinase (DNA-PK)-dependent manner. Integrated cellular, biochemical, and genomic analyses reveal an antagonistic yet correlated relationship between TOP2B and DNA-PK activities. Under normoxic conditions, TOP2B associates with HIGs and represses transcription by suppressing DNA negative supercoiling formation and accessibility. Under hypoxic exposure, TOP2B dissociates from HIGs, while DNA-PK and HIF1α are recruited to activate HIGs. Notably, DNA-PK is required for the repressive function of TOP2B, as DNA-PK knockout abrogates TOP2B release and activity, resulting in elevated expression of a number of HIGs. Mechanistically, DNA-PK phosphorylates TOP2B at T1403, stimulating its catalytic activity to restrain DNA unwinding and accessibility, thereby suppressing HIG transcription. Collectively, these findings identify a novel role for TOP2B and DNA-PK-mediated regulation of TOP2B as key transcriptional regulators of HIG expression. We propose that phosphorylation-dependent modulation of TOP2B activity is coordinated with transcriptional states and determines DNA topology to regulate Pol II transcription.

ABSTRACT Immediate early genes (IEGs) encode master transcription factors, including EGR1 , FOS , JUN , and MYC genes that drive robust transcription in the G1 phase of cell cycle. Our previous studies indicate that topoisomerase IIβ (TOP2B) critically regulates IEG transcription and that the activities of TOP2B are dynamically controlled through post-translational modifications by BRCA1-BARD1 and ERKs. Here, we show that two E2 enzymes, UBCH5b and UBC13/MMS2, differentiate the effects and functions of TOP2B ubiquitination by BRCA1-BARD1. Comprehensive transcriptomics, proteomics, and biochemical and molecular cellular analyses revealed a close relationship between BARD1 and key mitogen-activated protein kinase pathway genes and identified activated ERK2 as a novel kinase that phosphorylates BARD1 at S391, a previously reported mitotic phosphorylation site, whose genetic mutation has been linked to tumorigenesis. Mechanistically, the catalytic activity of ERK2 stimulates TOP2B ubiquitination mediated by BRCA1-BARD1 in complex with UBCH5b and UBC13/MMS2, which controls the binding and function of TOP2B and BARD1 for transcriptional activation at representative IEGs. Taken together, our data propose that there is a functional regulatory circuit involving TOP2B, BARD1, and ERK2, three key transcriptional activators for IEG transcription, in which the gene association and catalytic activity of TOP2B are regulated through E2-differentiated ubiquitination by BRCA1-BARD1 and the phosphorylation of BARD1 by ERK2 for productive transcription.

SV40 Large T antigen (LT) is essential for viral replication and a key determinant of host range. This host-range function is mediated by the C-terminal domain (LT-C) through binding to the host serine protease FAM111A, but the underlying mechanism has remained unclear. Here, we report the X-ray crystal structure of the FAM111A serine protease domain in complex with LT-C, revealing the structural basis for direct inhibition of FAM111A. LT-C uses a previously unrecognized zinc-binding motif and a P1-like phenylalanine residue to engage the FAM111A active site through a substrate-mimicking mechanism while avoiding proteolytic cleavage and covalent complex formation. Mutations disrupting either feature abolish FAM111A inhibition and impair SV40 propagation in cells. Consistent with this mechanism, SV40 host restriction requires FAM111A protease activity, which must be antagonized by LT-C for productive infection. Together, these findings define a zinc-dependent, cleavage-avoiding mechanism of protease inhibition that highlights an evolutionary arms race between SV40 and host antiviral proteases.

Protein kinase C (PKC) isozymes are ubiquitous kinases that direct diverse cellular pathways and are important drug targets for the treatment of cancer and neurological diseases. PKCs are auto-regulating enzymes governed by phospholipid and Ca2+ signals via a mechanism that has remained enigmatic due to a paucity of structural information. Herein we present a series of structures of the full-length human PKCβI and PKCβII isozymes. These structures reveal the molecular basis by which PKCs maintain an auto-inhibited state, convert to a defined and ordered active conformation via a "lipid-lever" mechanism of allosteric activation, and how isoform-specific differences alter their allosteric regulatory mechanisms. We show that endoxifen, a recently identified PKCβI inhibitor, can alter the allosteric regulatory mechanism of PKCβI, providing a proof of concept for allosteric regulators of PKCs. Collectively, our data describe a foundational molecular model of second messenger-mediated allosteric regulation of PKCs that underpins PKC function, misregulation, and mechanisms of inhibition.

Endocrine therapy (ET) with aromatase inhibitors (AI) plus ovarian function suppression (OFS) is standard for premenopausal women with ER+/HER2- breast cancer. However, many premenopausal women are intolerant of AI + OFS, leaving tamoxifen (with or without OFS) as their only option. Extensive studies have demonstrated that premenopausal women treated with neoadjuvant ET whose tumors exhibit Ki-67 ≤ 10% following 4 weeks of therapy (defined as endocrine sensitive disease (ESD)) achieve 5-year distant disease-free survival > 96%. Problematically, only 41% of patients reach this ESD threshold with tamoxifen compared to 78% with AI+OFS (Nitz JCO 2022). (Z)-endoxifen (ENDX) is a potent selective estrogen receptor modulator (SERM) that inhibits ERα at plasma concentrations of 3-5 ng/ml and dually targets PKCβ1, a suspected oncogene in breast tumors, at concentrations ≥ 500 ng/ml. We therefore developed the EVANGELINE study (NCT05607004) is an open label, randomized neoadjuvant phase II study that includes a pharmacokinetic (PK) Run-in, aiming to evaluate whether ENDX +/- goserelin is non-inferior to exemestane plus goserelin with respect to ESD rate. Following the completed PK run-in portion of the study, where 40mg/day or 80mg/day ENDX were assessed, the 40 mg/day dose was selected for further evaluation in the second part of the trial. The second part is comprised of a randomized treatment cohort, for patients with a Ki-67% >10% at preregistration randomized to either 40mg (Z)-endoxifen + goserelin or exemestane + goserelin neoadjuvant treatment and a Single Arm Treatment Cohort for patients who have a Ki-67 ≤ 10% at pre-registration assigned to 40mg/day (Z)-endoxifen monotherapy neoadjuvant treatment. To investigate ENDX further in premenopausal women, a multicenter study in the United States, was designed(EVANGELINE; NCT05607004). This phase 2, open-label, randomized, neoadjuvant trial includes a PK run-in phase designed to assess whether the 4-week ESD rate with (Z)-endoxifen + goserelin is non-inferior to that of exemestane + goserelin in premenopausal women with Stage IIA or IIB ER+/HER2- breast cancer with diagnostic Ki67 > 10%. Additionally, a single arm Phase 2, open-label neoadjuvant Cohort was designed to assess the 4-week ESD rate with ENDX monotherapy in premenopausal women with Stage IIA or IIB ER+/HER2- BC with diagnostic Ki67 ≤10%. Subjects will undergo neoadjuvant treatment for six months before receiving surgical treatment in the involved breast. This study will additionally assess safety, tolerability, response rates, surgical outcomes, and monitor disease progression. Comprehensive biomarker evaluations will be conducted using paired tumor biopsies and blood samples collected at baseline and week four, as well as 6 month surgical specimens. Enrollment in the PK run-in phase (n-22) was completed in Fall 2024 with the second portion of the trial slated to begin early in 2025. Matthew P. Goetz, Vera J. Suman, Claudia Lopez, Hayley Erickson, Lonnissa Nguyen, Sandra S. Hammer, Lida Mina, Pooja Advani, Roberto Leon-Ferre, Karthik Giridhar, Felipe Batalini, Daniel Flora, Jason M. Jones, Nusayba A. Bagegni, Katie N. Hunt, Mara Piltin, Amy Degnim, James N. Ingle, Judy C. Boughey, Sarah A. Buhrow, Joel M. Reid, Matthew Schellenberg, John R. Hawse, Steven C. Quay. A randomized phase 2 non-inferiority trial of (Z)-endoxifen + goserelin and exemestane + goserelin as neoadjuvant treatment for premenopausal women with ER+/HER2- breast cancer (EVANGELINE) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT237.

Topoisomerase II alpha and beta (TOP2A and TOP2B) isoenzymes perform essential and non-redundant cellular functions. Anthracyclines induce their potent anti-cancer effects primarily via TOP2A, but at the same time they induce a dose limiting cardiotoxicity through TOP2B. Here we describe the development of the obex class of TOP2 inhibitors that bind to a previously unidentified druggable pocket in the TOP2 ATPase domain to act as allosteric catalytic inhibitors by locking the ATPase domain conformation with the capability of isoform-selective inhibition. Through rational drug design we have developed topobexin, which interacts with residues that differ between TOP2A and TOP2B to provide inhibition that is both selective for TOP2B and superior to dexrazoxane. Topobexin is a potent protectant against chronic anthracycline cardiotoxicity in an animal model. This demonstration of TOP2 isoform-specific inhibition underscores the broader potential to improve drug specificity and minimize adverse effects in various medical treatments.

Breast cancer (BC) is the most common cancer diagnosed in American women. The majority of breast cancers express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER2). However, 15-20% lack expression of all three and are called triple-negative breast cancer (TNBC). TNBC accounts for a disproportionately large amount of morbidity and mortality due to its aggressive nature and the paucity of effective adjuvant maintenance therapy, driving an urgent need for novel therapeutics. Z-endoxifen (ENDX) is a potent tamoxifen metabolite that dually targets ERα and protein kinase C beta I (PKCβI). We recently described next-generation molecules, the first-in-class endoxifen-PROTAC (ENDX-PROTAC) compounds which incorporate ENDX as the warhead and cereblon (CRBN) as the E3 ubiquitin ligase recruitment moiety (Jayaraman 2024, SABCS). We used the PRISM screen of over 900 cancer cell lines to determine that ENDX-PROTACs have broad antitumor activity in both TNBC and ER+ breast cancer in addition to, leukemia, pancreatic, lymphoma, lung, melanoma, and ovarian cancer cells, among others. ENDX-PROTACs primarily target a key translation termination factor, G to S phase transition 1 (GSPT1). Here, we demonstrate for the first time that these compounds also cause marked DNA damage. The lead compounds of this series, EPrC-007 and EPrC-009 demonstrated potent anti-proliferative effects in MDA-MB-231, BT549, and MDA-MB-436 TNBC cell lines. EPrC-007 induced substantial DNA double strand breaks in MDA-MB-231 cells as assessed by γ-H2AX Western blotting. Interrogation of DNA repair pathways revealed activation of ATM/CHK2 and downregulation of ATR/CHK1, suggesting inhibition of DNA repair as a potential mechanism. Additionally, EPrC-007 also induced γ-H2AX foci in MDA-MB-231 and BT549 cells, but not in the MCF10A normal breast tissue cell line, suggesting selective anti-tumor activity. Since we suspect that EPrC-007 blocks DNA repair machinery, we next sought to determine if these molecules would synergize with DNA damaging agents or other DNA repair inhibitors. Promisingly, EPrC-007 demonstrated remarkable synergy with olaparib (poly-(ADP-ribose) polymerase (PARP) inhibitor), hydroxyurea (ribonucleotide reductase inhibitor), and cisplatin (direct DNA damaging agent), but not camptothecin (topoisomerase I inhibitor). Importantly, only olaparib co-treatment demonstrated synergy in the PARP- and PROTAC-resistant HCC1143 TNBC cell line. Furthermore, the combination of EPrC-007 with olaparib induced markedly more γ-H2AX foci than either agent alone, confirming synergy at the DNA damage level. Taken together, our studies demonstrate the remarkable potential of ENDX-PROTACs in the single-agent setting as well as in combination with PARP inhibitors. Aamod S. Dekhne, Swaathi Jayaraman, Lucy Rai Thulung, Becky Bruinsma, Baustin M. Welch, Samuel Wyatt, Esther P. Rodman, Sayantani Bhattacharya, Thomas R. Caulfield, John R. Hawse, Matthew J. Schellenberg, Matthew P. Goetz. Novel endoxifen-based proteolysis targeting chimera (PROTAC) small molecules synergize with olaparib and exhibit potent antitumor activity in triple negative breast cancer through induction of DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1666.

Recombinant human Topoisomerase 2 (TOP2) alpha and TOP2 beta proteins are valuable reagents for structural and biochemical analyses. Here, we describe a scalable method for production of high-purity recombinant TOP2 proteins using a HEK293F cell expression system with transient transfection of expression constructs. Human TOP2 is expressed as a fusion with YFP or mCherry, which serves dual purposes as an indicator of protein expression and an affinity tag that enables rapid isolation of TOP2 from cell lysates. Subsequent FPLC polishing of the protein yields milligram-scale yields of high purity and active recombinant TOP2.

Background: Aromatase inhibition (AI) with ovarian function suppression (OFS) is standard for premenopausal women (PrW) with ER+/HER2- primary breast cancer (BC). However, > 40% are intolerant of OFS leaving tamoxifen (TAM) as their only option. In the neoadjuvant endocrine setting, PrW with endocrine sensitive disease (ESD) (week 4 Ki-67 ≤ 10%) have 5-year disease free survival > 97%; however, only 35% achieve ESD with TAM vs 76% with AI+OFS (Gluz SABCS 2023). Z-endoxifen (ENDX) is a selective estrogen receptor modulator targeting ERα at 3-5 ng/ml and PKCβ1 ≥ 500 ng/ml, the latter resulting in AKT inhibition and apoptosis (Jayaraman NPJ Breast Cancer). EVANGELINE (NCT05607004) is an open label, randomized neoadjuvant phase II study with a PK Run-in to evaluate whether ENDX is non-inferior to exemestane plus goserelin in terms of ESD rate. The PK Run-in was designed to identify an ENDX dose that targets both ERα and PKCβ1, defined as day 28-day ENDX steady state concentration (Css) > 500 ng/ml in ≥ 5/6 patients (pts), and to assess antitumor activity and toxicity. In the PK Run-in, pts received either ENDX 40 mg/day (as monotherapy) or 80 mg/day (+/- goserelin). Pts with ESD remained on ENDX for 6 cycles (24 weeks) followed by surgery. We reported (Goetz AACR 2024) that among 7 pts (age 28-51; median 46) enrolled at the 40 mg/day dose, the median ENDX 28-day Css was 264 ng/mL (range:180-377), and ESD rate was 86% (6/7 pts), with either week 4 Ki-67 remaining ≤ 10% (n=3) or decreasing to ≤ 10% (n=3) with 1 pt remaining >10%. Grade 2 or higher AEs included grade 2 amenorrhea (n=2), grade 2 hot flashes (n=1) and grade 2 hypertension (n=1). The 6 pts with ESD completed 24 weeks of ENDX followed by surgery. Here we present the surgical findings of these 6 pts including Cell Cycle Arrest (CCA), Residual Cancer Burden (RCB), as well as the preliminary 4 week ESD rate for the 80 mg/day +/- goserelin cohorts. Methods: All pts who started protocol treatment are included in the summarized results. Results: The 6 pts treated with 24 weeks of ENDX 40 mg/day underwent surgery with no complications. cT stage was T2 in 5 pts and T3 in 1 pt. Central review of pre- and week 24 breast MRIs using RECIST criteria found tumor diameter reductions of 15 to 100% resulting in 1 CR, 1 PR and 4 SD. Median pre-Ki67 was 11 (range: 4-33%). Surgical CCA (Ki67 ≤ 2.7%) rate was 67% (4/6). The remaining 2 pts had Ki-67 of 3% (n=1) or in the patient with a near pCR, too few cells to quantitate (n=1). RCB was 1 (n=1), II (n=3), and III (n=2). PEPI scores will be reported at the meeting. At the 80 mg/day dose, ten pts were randomized to either ENDX alone (n=5, age 44-53; median 46) or ENDX + goserelin (n=5, age 37-52; median 44) with similar ER expression (all > 80%), cT, cN category and tumor grade in each arm. Ki-67 testing was completed pre- and week 4 for 9 pts where pre Ki-67 was ≤ 10% in 3 pts, > 10% in 4 pts, and not available in 2 pts (inadequate tumor cellularity). Following drug treatment, week 4 Ki-67 was maintained or fell to ≤ 10% in 6 pts, remained > 10% (1 pt), was indeterminate in 2 pts (inadequate tumor cellularity). PK data (80 mg/day) was available from the first 9 pts, and demonstrated a median 28 day ENDX Css of 344 ng/ml (range 161-959) with 3/9 pts > 500 ng/ml. At the 80 mg/day dose, there were no grade 3-4 toxicities. Grade 2 toxicities included hot flashes (2 pts), dyspepsia (1 pt), amenorrhea (1 pt), oligomenorrhea (1 pt), and nausea (1 pt). No VTE events were reported. Conclusions: Neoadjuvant ENDX, when administered to PrW, demonstrated substantial antitumor activity when administered as monotherapy (40 or 80 mg/day) or with OFS (80 mg/day). Increasing the ENDX dose from 40 to 80 mg improved the likelihood of achieving an Endx Css > 500 ng/ml without significantly increasing toxicity. We will present the final results from the 80 mg/day cohort at the meeting. Citation Format: Matthew Goetz, Vera J. Suman, Joel M. Reid, Lida A. Mina, Pooja Advani, Arezoo Mirad, Roberto Leon Ferre, Karthik Giridhar, Felipe Batalini, Sarah Buhrow, Stephanie Safgren, Swaathi Jayaraman, Patricia Cronin, Mara Piltin, Amy C. Degnim, Sarah Premji, James N. Ingle, Tufia Haddad, Amye J. Tevaarwerk, Jason Jones, Daniel Flora, Harjinder Singh, Nusayba Bagegni, Katie N. Hunt, Judy C. Boughey, Matthew Schellenberg, John Hawse, Steven C. Quay. Neoadjuvant Z-endoxifen for Premenopausal Estrogen Receptor (ER)+, Human Epidermal Growth Factor Receptor (HER2)- Breast Cancer (BC): Evaluation of the Pharmacokinetic (PK) Run-in for the EVANGELINE Study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-11-04.

Background: Premenopausal women (PrW) with ER+, HER2- breast cancer (BC) are commonly treated with aromatase inhibitors (AI) with ovarian function suppression (OFS) or tamoxifen (tam) +/- OFS. Common adverse effects (AE) include musculoskeletal symptoms, hot flashes, night sweats, mood changes, and sexual side effects that may seriously impact quality of life (QOL). In SOFT, compared to tam alone, pts treated with tam + OFS had more hot flashes, loss of sexual interest, sleep disturbances, and vaginal dryness. In the combined SOFT/TEXT analysis, pts receiving exemestane + OFS had greater bone/joint pain, vaginal dryness, and loss of sexual interest, whereas pts receiving tam + OFS had more hot flushes and sweats. In the premenopausal setting, E1/E2 levels differ based on type of endocrine therapy regimen, with menopausal E1/E2 levels following OFS, but increases with tam monotherapy. EVANGELINE (NCT05607004) is an ongoing phase 2 multicenter neoadjuvant study assessing (Z)-endoxifen (ENDX), a potent tam metabolite that dually targets ERα and PKCβ, in PrW with ER+/HER2- BC. Prior to the randomized phase II, a PK run-in was designed to identify a dose wherein 28-day ENDX Css > 500 ng/ml in ≥ 5/6 pts and to assess antitumor activity and toxicity when ENDX is dosed at 40 mg/day (monotherapy) and 80 mg/day (+/- OFS). Pts with endocrine sensitive disease (ESD) defined as 4-week Ki67 ≤ 10% continue treatment (tx) for 24 weeks followed by surgery. Here, we report the menopausal symptoms for pts enrolled onto 40 mg/day cohort. Methods: All eligible pts who completed baseline and at least one tx questionnaire or toxicity evaluation are included. Data captured by the medical team using Common Terminology Criteria for Adverse Events (CTCAE) and self-reported symptoms are reported in pts menstrual diary or on the Menopause-Specific Quality of Life (MENQOL) questionnaire administered at baseline, end of week 4, 12, and 24. MENQOL bothersomeness level was categorized as mild (score 0-1), moderate (2-4), and high (5-6). Estradiol and estrone levels were assessed for the 40 mg/day dose at week 4 and 24. Results: For the 40 mg/day cohort, 7 PrW (6 White, 1 Asian) aged 28-51 (median 46) received ENDX. One pt discontinued due to week 4 Ki-67 > 10%. The remaining 6 had ESD, and after 24 weeks underwent surgery. Over the course of tx, relevant AEs reported as possibly, probably or definitely attributed to ENDX included: amenorrhea (G2-n=5); dysmenorrhea (G2-n=1; G1-n=1), hot flashes (G2-n=1; G1-n=5), hyperhidrosis (G1-n=1), hypomenorrhea (G1-n=1), irregular menstruation (G1-n=2), and libido decrease (G2-n=1). Menses did not occur on tx for 4 pts. No dose reductions occurred. Among the remaining 3 pts, dysmenorrhea was described as mild/moderate. MENQOL after 4 wks of tx revealed that hot flashes developed in 4 pts (moderately bothersome -3 pts, not bothersome -1 pt). One pt began venlafaxine for hot flashes. Three pts reported ‘being impatient with others’ as moderately bothersome. The median (range) baseline estrone (n=5) was 54 pg/mL (19-114) with median (range) fold increase from baseline of 9.0 (1.3-23.2 pg/mL) at wk4 and 4.7 (0.4 - 25.9 pg/mL) at wk24. The median baseline estradiol level (n=5) was 29 pg/mL (19-209) with median fold increases from baseline of 17.9 (0.4-57.0 pg/mL) at wk4 and 8.1 (0.04 - 56.6 ng/mL) at wk24. Currently, 12 pts are enrolled to either 80 mg/day monotherapy or 80 mg/day + OFS. Conclusions: This is the first report of menopausal side effects in PrW women treated with ENDX 40 mg/day without OFS. Most side effects were low grade and amenorrhea was common. ENDX induced marked increases in E1/E2 levels that peaked at 4 weeks and then declined. MENQOL data from the 80 mg/day monotherapy and 80 mg/day + OFS cohort will be reported at the meeting. Citation Format: Sarah K. Premji, Vera J. Suman, Lida A. Mina, Pooja P. Advani, Arezoo Mirad, Roberto Leon-Ferre, Karthik V. Giridhar, Felipe Batalini, Swaathi Jayaraman, Patricia Cronin, Mara Piltin, Amy Degnim, James N. Ingle, Tufia C. Haddad, Amye J. Tevaarwerk, Jason M. Jones, Daniel Flora, Harjinder Singh, Nusayba A. Bagegni, Katie N. Hunt, Judy C. Boughey, Joel M. Reid, Matthew Schellenberg, John R. Hawse, Steven Carl Quay, Matthew P. Goetz. Neoadjuvant (Z)-endoxifen for Premenopausal Estrogen Receptor (ER)+, Human Epidermal Growth Factor Receptor 2 (HER2)- Breast Cancer (BC): Evaluation of Quality of Life (QOL) measures in the EVANGELINE Study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-03-07.

Background: In the estrogen receptor positive (ER+) breast cancer, the tamoxifen metabolite Z-endoxifen (ENDX) potently inhibits ERα and partially degrades protein kinase C beta I (PKCβI) resulting in suppression of AKT signaling and the induction of apoptosis (Jayaraman et al, NPJ Breast Cancer, 2023). Intriguingly, at higher concentrations (5 µM and higher), ENDX inhibits the viability of triple negative breast cancer (TNBC) cells and multiple non-breast cancer and ER- cell line models. We thus surmised that ENDX may elicit its anti-cancer activity through mechanisms extending beyond inhibition of ER and PKCβI, and that development of more potent ENDX derivatives would extend the clinical utility of this molecule. Therefore, we developed a series of chemically synthesized ENDX-PROTAC (EPrC) compounds with ENDX as the warhead and cereblon (CRBN) as the E3 ubiquitin ligase recruitment moiety with the expectation that degradation of ENDX targets would elicit more potent anti-cancer activity at lower concentrations. Methods: We characterized the anti-cancer activity of multiple EPrC compounds using multiple ER+ and TNBC cell line models and performed a series of unbiased approaches to identify novel proteins for which ENDX is a substrate. Results: Initial studies in ER+/HER2- breast cancer cell lines showed that EPrC’s resulted in no greater anti-cancer effects compared to ENDX. However, three EPrC’s (EPrC 7-9) exhibited profound anti-cancer activity at nanomolar concentrations in chemosensitive (MDAMB231, BT549, MDAMB436 and MDAMB468) as well as chemoresistant (doxorubicin-resistant and paclitaxel-resistant MDAMB231) TNBC cells. Conversely, ENDX had little activity in these models even at 5 µM concentrations. The potent anti-cancer activity of EPrC 7-9 also extended to chemoresistant TNBC patient-derived organoid (PDO) models. In contrast, EPrC 7-9 displayed no effects on proliferation or apoptosis in the non-malignant MCF10A breast epithelial cell line. We further evaluated the anti-cancer activity of EPrC’s and ENDX using the NCI-60 human cancer cell line panel where broad anticancer activity was additionally observed in leukemia, lung, melanoma, and ovarian cancer cells. Mechanistically, EPrC 7-9 activity was independent of PKCβ1 expression/targeting. Therefore, we performed an unbiased SILAC and TMT-based mass spectrometry (MS) analysis utilizing MDAMB231 and MCF10A cells to identify EPrC target proteins that are ubiquitinated and degraded in MDAMB231 cells but not MCF10A cells. One of the top hits from this screen was G to S phase protein 1 (GSPT1), a conserved protein that regulates protein translation termination. We validated GSPT1 as a bona fide and rapidly targeted protein by EPrC’s with near complete GSPT1 elimination occurring within four hours of treatment. The necessity of EPrC-mediated GSPT1 degradation to the anti-cancer activity of these molecules was confirmed using CRBN knockout MDAMB231 cells where EPrC 7-9 completely failed to inhibit cell proliferation, induce apoptosis, or alter GSPT1 protein levels. Conclusion: Taken together, we have developed a new class of PROTAC drugs with ENDX as the warhead, which display potent anti-cancer activity in chemosensitive and chemoresistant models of TNBC, as well as non-breast cancer cell lines, and implicate GSPT1 as a novel and direct target of ENDX. Efforts are ongoing to elucidate the structural basis of EPrC-GSPT1 interaction, to assess GSPT1’s contribution to EPrC activity and to study the pharmacology, toxicity, and in vivo antitumor activity of these molecules. Citation Format: Swaathi Jayaraman, Sayantani Sarkar Bhattacharya, Thomas Caulfield, Stephanie L. Safgren, Sarah A. Buhrow, Joel M. Reid, Mary J. Kuffel, Jasmine K. Farmakes, Akhilesh Pandey, James N. Ingle, Matthew M. Ames, John R. Hawse, Matthew J. Schellenberg, Matthew P. Goetz. Z-Endoxifen-PROTAC, a novel drug for chemo-sensitive and-resistant Triple Negative Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-12-23.

We identified small molecule tricyclic pyrone compound CP2 as a mild mitochondrial complex I (MCI) inhibitor that induces neuroprotection in multiple mouse models of AD. One of the major concerns while targeting mitochondria is the production of reactive oxygen species (ROS). CP2 consists of two diastereoisomers, D1 and D2, with distinct activity and toxicity profiles. This study was designed to understand how structure of D1 and D2 affects their binding to MCI and the consequential impact on ROS production. The X-ray crystallography and cryo-electron microscopy (cryo-EM) at global resolution of 3.25-3.27Å were employed to identify the molecular structure of D1 and D2 and the D1 binding to the isolated ovine MCI. The assessment of the MCI inhibition and the extent of ROS generation were done in isolated MCI and human neuroblastoma MC65 cells using flow cytometry, a Seahorse extracellular flux analyzer, and the kinetic studies. In the closed conformation of MCI, D1 selectively binds to the deep Quinone-site (Q d ) but not to the shallow Q-site (Q s ), sharing the same binding pocket as rotenone. In the open MCI state, D1 exclusively binds to the Q s in contrast to rotenone, which binds Q d and Q s in both closed and open states. At the same concentrations, D1 inhibits respiration to a greater extent compared to D2 (5:1 ratio) and produces higher level of ROS. Cryo-EM unambiguously identified binding of D1 to both the Q d and Q s sites, contingent upon the conformational state of MCI. In contrast to rotenone, D1 binds Q d only in the closed conformation during catalytic cycle, leading to mild inhibition. Superimposing X-ray crystallography data of D1 and D2 onto cryo-EM data suggests that the orientation of the methyl group in D2 induces a flatter conformation, resulting in lower binding affinity to MCI, which correlates with lower inhibition and toxicity compared to D1. At physiologically relevant concentrations, CP2 (D1:D2 = 1:1) demonstrates low MCI inhibition yielding negligible ROS levels. This observation provides new insight into the absence of toxicity associated with CP2 treatment in vivo , further highlighting feasibility for the development of safe and efficacious MCI inhibitors.

The host-range mutant of rabbitpox virus (RPXV) with a deletion in the gene encoding the serpin serine protease inhibitor 1 (SPI-1) fails to replicate efficiently in restrictive host cells. Depletion of the host cell serine protease FAM111A restores viral replication in these cells, suggesting that SPI-1 targets FAM111A to facilitate infection. However, direct evidence of SPI-1 inhibiting FAM111A has been lacking. Here, we demonstrate that SPI-1 directly inhibits FAM111A's protease activity in vitro through covalent complex formation, a hallmark of the serpin inhibition mechanism. SPI-1 also exhibits specificity for FAM111A compared to other serine proteases in vitro. Through mutagenesis studies, we identified residues and regions within SPI-1's reactive center loop (RCL) that are critical for FAM111A inhibition and covalent complex formation in vitro, with varying degrees of impact. Notably, these RCL mutations showed a spectrum of effects on SPI-1's ability to support RPXV replication in non-permissive cells, which strongly correlated with their impact on SPI-1's capacity to inhibit FAM111A activity in vitro. Altogether, our study provides direct evidence that SPI-1 inhibits FAM111A protease activity, highlighting FAM111A's antiviral role and its significance as a target of SPI-1 during orthopoxvirus infection.

Anthracyclines are potent chemotherapeutic agents, yet their clinical use is hampered by their cardiotoxicity. Anthracyclines achieve potent anti- cancer effects primarily via TOP2A, but at the same time they induce a dose limiting cardiotoxicity through TOP2B. The use of a catalytic TOP2 inhibitors such as dexrazoxane (ICRF 187) significantly reduces the incidence of anthracycline-induced cardiotoxicity. However, dexrazoxane targets the highly conserved TOP2 dimer interface that is identical between TOP2A and TOP2B, fostering persisting concerns that its inhibition of TOP2A isoenzyme may interfere with anthracycline anticancer effects and augment anthracycline-induced myelosuppression. Thus, isoform- selective TOP2 inhibitors that can control the DNA damage caused by TOP2A vs TOP2B are a significant unmet clinical need.We have developed a new class of highly potent and TOP2B-selective catalytic inhibitors through rational design by building upon the general pharmacophore present in isolates from Calophyllum Brasiliense. Using X-ray crystallography, we demonstrate that our newly developed compounds occupy a previously unidentified druggable binding-site in the TOP2 ATPase domain and function as a physical barrier to block the conformational change triggered by ATP hydrolysis. We have named the class of inhibitors that target this site “obex”, the Latin word for obstacle. The obex binding site contains residues that differ between the TOP2A and TOP2B isoenzymes, based on which we rationally designed, prepared, and systematically evaluated a targeted panel of obex inhibitors. We have identified the lead molecule, namely, topobexin, as the first- in-class TOP2B-specific obex inhibitor that can prevent anthracycline-induced DNA damage. This first TOP2 isoform-specific inhibition underscores the broader potential to improve drug specificity and minimize adverse effects in various medical treatments.

Background: Ovarian function suppression (OFS) when combined with tamoxifen or an aromatase inhibitor is the standard of care for premenopausal women (preMW) with estrogen receptor positive (ER+) human epidermal growth factor receptor 2 negative (HER2-) breast cancer (BC) based on SOFT (Francis 2022) and TEXT (Pagani 2022) clinical trials and confirmed in a metanalysis (EBCTCG 2022). However, estrogen deprivation in preMW is associated with significant morbidity and up to 40% of premenopausal women with ER+/HER2- BC are intolerant of OFS. For these women, tamoxifen monotherapy is the only FDA approved option where long-term patient outcomes are known to be sub-optimal. Therefore, there is substantial room to improve endocrine therapy for preMW with ER+/HER2- BC. (Z)-endoxifen is one of the most active anti-estrogen tamoxifen metabolites, with preclinical data demonstrating superiority to tamoxifen and letrozole in aromatase expressing xenograft models and evidence for antitumor activity in early phase clinical trials in patients with prior progression on endocrine therapy including tamoxifen. In addition to its ability to potently block ERα, (Z)-endoxifen at clinically achievable concentrations inhibits protein kinase C beta 1 (PKCβ1), resulting in downstream AKT inhibition and apoptosis. It is hypothesized that (Z)-endoxifen, when delivered as monotherapy at doses that dually target both ERα and PKCβ1, will potently inhibit BC proliferation while obviating the need for OFS in premenopausal women. If successful, this innovative approach could spare women with endocrine sensitive BC from the side effects of OFS. Methods: To further study (Z)-endoxifen in preMW, a multicenter study in the United States, with possible expansion to other countries was designed (EVANGELINE; NCT05607004). This is a Phase 2, open-label, randomized, neoadjuvant study with a pharmacokinetic (PK) run-in phase in preMW with Stage IIA or IIB ER+/HER2- incident BC. Subjects are randomized 1:1 to either exemestane plus goserelin or (Z)-endoxifen monotherapy. The primary objective of the PK-run is to determine the dose that inhibits PKCβ1 and in the randomized Phase II is to determine whether the endocrine sensitive disease (ESD) rate (defined as Ki-67 ≤ 10% after 4 weeks of neoadjuvant therapy) with (Z)-endoxifen is non-inferior to exemestane plus goserelin. Subjects will be enrolled with the intent of surgical treatment in the involved breast after completing 6 months of neoadjuvant treatment. Safety, tolerability, response rates and surgical outcomes will be assessed. Disease progression will be monitored throughout study participation. Paired tumor biopsies and blood sampling will allow for a robust biomarker evaluation. Enrollment to the 40mg PK Run-was completed in June 2023 (n=7), and accrual to the 80mg PK Run-in will be complete by August 2024. The randomized portion of the trial is expected to commence in Fall 2024. Citation Format: Matthew Goetz, Vera J. Suman, Arezoo Mirad, Harjinder Singh, Sandra S. Hammer, Lida Mina, Pooja Advani, Roberto Leon-Ferre, Karthik Giridhar, Felipe Batalini, Daniel Flora, Jason M. Jones, Nusayba A. Bagegni, Katie N. Hunt, James Jakub, Mara Piltin, Amy Degnim, James N. Ingle, Judy C. Boughey, Sarah A. Buhrow, Joel M. Reid, Matthew Schellenberg, John R. Hawse, Steven C. Quay. A Randomized Phase 2 Non-inferiority Trial of (Z)-endoxifen and Exemestane + Goserelin as Neoadjuvant Treatment for Premenopausal Women with ER+/HER2-Breast Cancer (EVANGELINE) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-12-18.

The open reading frame 8 (ORF8) protein, encoded by the SARS-CoV-2 virus after infection, stimulates monocytes/macrophages to produce pro-inflammatory cytokines. We hypothesized that a positive ex vivo monocyte response to ORF8 protein pre-COVID-19 would be associated with subsequent severe Coronavirus disease 2019 (COVID-19). We tested ORF8 ex vivo on peripheral blood mononuclear cells from 26 anonymous healthy blood donors and measured intracellular cytokine/ chemokine levels in monocytes by flow cytometry. The percentage of positive monocyte staining in the sample and change in mean fluorescence intensity (ΔMFI) after ORF8 were used to calculate the adjusted MFI for each cytokine. We then tested pre-COVID-19 peripheral blood mononuclear cell samples from 60 chronic lymphocytic leukemia (CLL) patients who subsequently developed COVID-19 infection. Severe COVID-19 was defined as hospitalization due to COVID-19. In the 26 normal donor samples, the adjusted MFI for interleukin (IL)-1β, IL-6, IL-8, and CCL-2 were significantly different with ORF8 stimulation versus controls. We next analyzed monocytes from pre-COVID-19 PBMC samples from 60 CLL patients. The adjusted MFI to ORF8 stimulation of monocyte intracellular IL-1β was associated with severe COVID-19 and a reactive ORF8 monocyte response was defined as an IL-1β adjusted MFI ≥0.18 (sensitivity 67%, specificity 75%). The median time to hospitalization after infection in CLL patients with a reactive ORF8 response was 12 days versus not reached for patients with a non-reactive ORF8 response with a hazard ratio of 7.7 (95% confidence interval: 2.4-132; P=0.005). These results provide new insight on the monocyte inflammatory response to virus with implications in a broad range of disorders involving monocytes.

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

Abstract The protein kinase C (PKC) family of enzymes are indispensable serine-threonine kinases involved in signal pathways implicated in many different cellular processes. Dysregulation of PKC isoforms may contribute to the development and progression of breast cancer. Z-Endoxifen (Z-ENDX), the active metabolite of tamoxifen (TAM) and a selective estrogen receptor modulator (SERM) exhibited potent antitumor activity in endocrine-resistant hormone receptor-positive breast and other gynecologic cancers. Mechanistically, we discovered that ENDX inhibits PKCβI and downstream AKT signaling. Based on insights from our recently determined crystal structure of PKCβI, we investigated the effects of ENDX on PKCβI in estrogen receptor-positive (ER+) breast cancer. In vitro FRET-based biochemical assays revealed that ENDX inhibits the kinase activity of conventional (PKCβI) as well as a novel (PKC;) PKC isoform with a similar IC50. However, the PKCβI catalytic domain was found to be less sensitive to ENDX compared to full-length PKCβI. We identified a multi-domain mechanism of PKC inhibition through an allosteric inhibitory model using X-ray crystallography. Since the spatial assembly of PKC is crucial for its kinase activity, we assessed the effects of ENDX on the intracellular localization of PKCβI using live cell confocal imaging. Sub-cellular tracking of YFP-tagged PKCβI in ER+ MCF7 cells paradoxically revealed that ENDX promoted the translocation of PKCβI to the plasma membrane in both a dose and time-dependent manner. Furthermore, co-treatment with inhibitors of PHLPP phosphatases, which regulate PKC, alleviated this translocation. Based on our prior data demonstrating greater antitumor activity of the Z vs E isomer of ENDX, we discovered that Z-ENDX inhibits PKCβI kinase activity more effectively than its E-isomer using in vitro kinase assays. Further, E-ENDX failed to promote PKCβI translocation to the membrane.Taken together, these results suggest that allosteric effects of Z-ENDX trigger PKCβI recruitment to the plasma membrane where it is normally active yet suppresses its kinase activity. These findings indicate that Z-ENDX induces a non-productive structure of the enzyme and "breaks" the well-known mechanism of PKC activation upon binding to the cell membrane. Moreover, Z-ENDX likely triggers PHLPP-mediated PKCβI dephosphorylation, and ultimately PKCβI degradation, suggesting that Z-ENDX represents a new mechanistic basis for targeting and downregulating PKCβI, and potentially other PKC family members, in cancer cells. This newly discovered function of Z-ENDX likely contributes to its superior anti-cancer effects when compared to TAM and other endocrine therapies. Citation Format: Sayantani Sarkar Bhattacharya, Huy V. Huynh, Jasmin K. Farmakes, Taylor L. Witter, Elizabeth S. Bruinsma, Swaathi Jayaraman, Anh T. Cong, John R. Hawse, Matthew P. Goetz, Matthew J. Schellenberg. Protein kinase C beta 1 (PKCβ1) is allosterically inhibited and paradoxically translocated to the membrane by z-endoxifen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB051.